Optimization of mESC transfection.

<p><b>A.</b> pCas9-GFP expression plasmid was transfected into mESCs by the indicated method. GFP fluorescence was assessed by fluorescent microscopy and FACS. Numbers indicate the fraction of GFP-expressing cells. <b>B.</b> Percent of transfected cells and mean fluorescence intensity of GFP+ cells. n = 3. Graphs show mean ± s.e.m.</p

Effect of homology arm length for Cas9-mediated knockin.

<p><b>A.</b> GFP knockin at Oct4 C-terminus using 50 bp or 200 bp homology arms. Knockin efficiency was measured by FACS for GFP expression. <b>B–C.</b> GFP expression in GFP⁺ FACS clones by microscopy and western blotting. <b>D.</b> PCR genotyping of individual ESC clones. The PCR product (arrowhead) is diagnostic of knockin at <i>Oct4</i>.</p

Knockout of 3 congenital heart disease genes in mESCs.

<p><b>A.</b> Experimental outline. Paired gRNAs are designed to induce a deletion of about 100 bp. <b>B.</b> PCR genotyping of pooled ESC genomic DNA after <i>Smad2</i> targeting. The mutant PCR product predominated. <b>C.</b> Surveyor nuclease on pooled ESC genomic DNA. Red arrowheads indicate nuclease cleavage products indicative of heterodimers. <b>D.</b> PCR genotyping of individual clones showing Smad2^Δ/Δ, Smad2^+/+, and Smad2^+/Δ clones. <b>E–F.</b> Frequency of <i>Smad2</i> genotypes amongst clonal outgrowths, after enrichment for transfected cells by GFP FACS or by transient puromycin selection. <b>G.</b> Western blot measurement of <i>Smad2</i> expression in wild-type and Smad2^Δ/Δ clones. <b>H.</b> Frequency of <i>Mll2</i> genotypes amongst 39 genotyped clones. <b>I.</b> Confirmation of <i>Mll2</i> inactivation in individual Mll2^Δ/Δ clones by qRTPCR. <b>J.</b> Frequency of <i>Chd7</i> genotypes amongst 48 genotyped clones. <b>K.</b> Confirmation of <i>Chd7</i> inactivation in individual Chd7^Δ/Δ clones by qRTPCR.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Cas9 gene knockout using single or dual gRNAs.

<p><b>A.</b> mESC cell line L10a-GFP, in which one Rosa26 locus expresses GFP. <b>B.</b> Experimental outline and gRNAs. gRNAs #9 and #5 generate 5′ overhangs for dual nickase strategy. <b>C.</b> Single gRNA-directed GFP inactivation. <b>D.</b> Comparison of gRNA alone or gRNA+HDR donor containing a translational stop signal. n = 3. Bar = s.e.m. Red circles indicate individual data points. <b>E.</b> Gene inactivation frequency of single compared to paired gRNAs. <b>F–G.</b> Assessment of GFP expression in mESCs in the GFP-FACS fraction by fluorescent microscopy and western blotting. <b>H.</b> Effective gene knockout using dual gRNAs and the hCas9-D10A nickase mutant.</p

Cas9-mediated introduction of a single base change.

<p><b>A.</b><i>Sap130</i> targeting strategy. The blue arrow indicates the gRNA targeting the reverse strand with the tail and tip representing the 5′ and 3′ ends, respectively. The HDR donor oligo is indicated by the green arrows. The T>C change is indicated in red. <b>B.</b> Surveyor nuclease assay on pooled ESC genomic DNA. Red arrowheads indicate nuclease cleavage products, diagnostic of heterodimers. <b>C.</b> Distribution of <i>Sap130</i> mutations. <b>D.</b> Sequence chromatograms showing homozygous T>C mutation.</p